ES2407859T3 - Multimeric constructions. - Google Patents

Abstract

A fusion protein of formula X-Y-Z, wherein: X comprises a portion of the VEGF receptor; And it is a linker moiety; and Z is a multimerization domain, wherein the linker moiety Y is a polypeptide of 5-50 amino acid residues that has an above-average flexibility as determined by the method of predicting flexibility of Karpus and Schultz, 1985, Naturwiss, 72: 212-213, and where said fusion protein binds to VEGF when multimerized.

Description

Multimeric constructions.

Field of the Invention

The invention relates to recombinantly constructed proteins useful for treating pathological neovascularization, for example, asthma, arthritis, cancer, and macular degeneration.

Background of the invention

Pathological neovascularization is a key component of diseases such as wet age-related macular degeneration (AMD), proliferative diabetic retinopathy, rheumatoid arthritis, osteoarthritis, and asthma. It also plays an important role in the growth and spread of tumors. Neovascularization is regulated by a delicate balance of pro- and anti-angiogenic factors.

It is known that vascular endothelial growth factor (VEGF) is necessary for neovascularization. It has been shown that inhibition of VEGF activity inhibits neovascularization in animal models of AMD, arthritis and in various tumor models. The methods used to inhibit VEGF activity include antibodies, receptor fusion proteins, peptides and small molecules.

VEGF-R1 (Flt-1) and VEGF-R2 (KDR) proteins have been shown to bind VEGF with high affinity. Both Flt-1 and KDR have seven Ig-like domains in their extracellular region. Domino 2 has been shown to be essential for VEGF binding. Fusions of each of the soluble full-length receptors (domains 17) and domains 1-3 with IgG Fc bind to VEGF efficiently. Fc fusions of IgG with the type Ig 2 domain alone, however, were unable to bind to VEGF, since they were a combination of type Ig 1 and 2 domain. Davis-Smyth, 1996. Therefore, type Ig domains. 1 and 3 appear to be necessary along with domain 2 for effective launching to VEGF.

Holash et al. (Proceedings of the National Academy of Science, vol. 99 (17): 11393-11398 (2002)) describe additional fusion polypeptides comprising portions of the VEGF-R1 receptor fused to the Fc part of the heavy chain of IgG1.

WO 00/75319 describes polypeptides that are modified chimeric Flt1 receptors and are said to have an improved pharmacokinetic profile.

WO 97/44453 describes proteins that are chimeric VEGF receptors and are said to bind to VEGF and quentagonize their angiogenic and proliferative activity of endothelial cells.

Olafsen et al. (Protein Engineering, Design & Selection, v. 17 (4), p. 315-323 (2004)) describes the characterization of anti-p185HER-2 (scFv-CH3) 2 antibodies constructed by genetic engineering.

Hu et al. (Cancer Research, v. 56 (13), p. 3055-3061 (1996)) describes a genetically engineered anti-antigen-carcinogen antibody (scFv-CH3) fragment and is said to have rapid and precise targeting of xenografts.

Brief summary of the invention

In accordance with one aspect of the present description, a fusion protein is provided. The fusion protein has the formula X-Y-Z. X comprises a polypeptide selected from the group consisting of a rextracellular receptor, a variable antibody region, a cytokine, a chemokine, and a growth factor. And essentially a polypeptide of 5-25 amino acid residues. Z is a CH3 region of an IgG heavy chain molecule.

Another aspect of the present description is a polypeptide of formula X-Y-Z. X comprises a polypeptide selected from the group consisting of an extracellular receptor, a variable antibody region, a cytokine, a chemokine, and a growth factor. And it consists essentially of a linker moiety that provides the spatial separation of 5-25 amino acid residues. Z is a CH3 region of an IgG heavy chain molecule.

Another aspect of the present description is a fusion protein of formula X-Y-Z. X comprises a polypeptide selected from the group consisting of an extracellular receptor, a variable antibody region, a cytokine, a chemokine, and a growth factor. And it consists essentially of a polypeptide of 5-25 amino acid residues. Z is an Fc part of an antibody molecule.

The present description also provides a fusion protein of formula X-Y-Z. X comprises a polypeptide selected from the group consisting of an extracellular receptor, a variable antibody region, a cytokine, a chemokine, and a growth factor. And it consists essentially of a linker moiety that provides the spatial separation of 5-25 amino acid residues. Z is an Fc part of an antibody molecule.

Another aspect of the present description is a method for multimerizing an X polypeptide. An X polypeptide is linked to a Z polypeptide by a Y polypeptide to form the XYZ polypeptide. X comprises a polypeptide selected from the group consisting of an extracellular receptor, a variable antibody region, a cytokine, a chemokine, and a growth factor. And it consists essentially of a polypeptide of 5-25 amino acid residues. Z is a CH3 region of an IgG heavy chain molecule. The XYZ polypeptide that forms semi-polymerizes.

Another aspect of the present description provides a method for multimerizing an X polypeptide. The X polypeptide is linked to a Z polypeptide by a Y moiety to form the XYZ polymer. X comprises a polypeptide selected from the group consisting of an extracellular receptor, a variable antibody region, a cytokine, a chemokine, and a growth factor. And it consists essentially of a linker moiety that provides the spatial separation of 5-25 amino acid residues. Z is a CH3 region of an IgG heavy chain molecule. The XYZ polypeptide thus formed is multimerized.

In one aspect of the present description a nucleic acid is provided. The nucleic acid molecule encodes a fusion protein comprising an Ig 2 type domain of VEGF-R1 (Flt-1); a linker; and a demultimerization domain. The fusion protein comprises a sequence selected from the group consisting of SEQ ID NO. 2, 8, 21, 23, and 25.

In another aspect of the present description a fusion protein is provided. The fusion protein comprises an Ig 2 type domain of VEGF-R1 (Flt-1), a linker, and a multimerization domain. The fusion protein comprises a sequence selected from the group consisting of SEQ ID NO: 2, 8, 21, 23, and 25.

In another aspect of the present description an in vitro method is provided. A nucleic acid molecule is supplied to an isolated mammalian cell. The nucleic acid molecule encodes a fusion protein that comprises an Ig 2 type domain of VEGF-R1 (Flt-1); a linker; and a multimerization domain. The defusion protein comprises a sequence selected from the group consisting of SEQ ID NO: 2, 8, 21, 23, and 25. The expression of the fusion protein is controlled by a promoter. A cell that expresses a fusion protein is formed.

Another aspect of the present description is a method of delivering a fusion protein to a mammal. A mammalian cell expressing the fusion protein is supplied to a mammal. The cell expresses and secretes the fusion protein thereby supplying the fusion protein to the mammal. The fusion protein comprises an Ig 2 type domain of VEGF-R1 (Flt-1), a linker, and a multimerization domain. The defusion protein comprises a sequence selected from the group consisting of SEQ ID NO: 2, 8, 21, 23, and 25.

Another aspect of the present description is a method of delivering a fusion protein to a mammal. It supplies a fusion protein comprising an Ig 2 type domain of VEGF-R1 (Flt-1), a linker, and a multimerization domain to a mammal. The fusion protein comprises a sequence selected from the group consisting of SEQ ID NO: 2, 8, 21, 23, and 25. Alternatively, a nucleic acid construct encoding said fusion protein can be supplied to the mammal, whereby the mammal Expresses protein defusion.

These and other aspects of the present description that will be described in more detail below provide the technique with methods and agents for treating diseases related to vascular proliferation and inflammation. Agents can provide increased stability and bioavailability in relation to the natural forms of proteins.

Now a part of the description that is contained in this document provides the aspects and embodiments of the present invention. More particularly, a first aspect of the present invention provides a fusion protein of formula XYZ, in which: X comprises the Ig 2 type domain of VEGF-R1 but lacks the Ig 1 and 3 type domains of VEGF-R1, said domain being type Ig 2 of VEGF-R1 covalently linked to the Z moiety through the Y moiety; And it consists of a polypeptide of 5-25 amino acid residues; and Z is a CH3 region of an IgG heavy chain molecule or an Fc part of an antibody molecule.

In a related aspect, the present invention provides a composition comprising this defusion protein of the invention, said composition further comprising one or more pharmaceutically acceptable excipients or carriers.

In a further aspect, the present invention provides a method for multimerizing an X polypeptide, the method comprising: attaching an X polypeptide to a Z polypeptide by a Y polypeptide to form an XYZ polypeptide, wherein: X comprises the Ig 2 type domain of VEGF-R1 but lacks VEGF-R1 Ig 1 and 3 type domains, said VEGF-R1 Ig 2 type domain being covalently linked to polypeptide Z by polypeptide Y; And it consists of a polypeptide of 5-25 amino acid residues; and Z is a CH3 region of an IgG heavy chain molecule or an Fc part of an antibody molecule; whereby the XYZ polypeptide multimerizes.

In further aspects, the present invention provides a nucleic acid molecule encoding the fusion protein of the present invention, a vector comprising said nucleic acid molecule, a mammalian cell comprising the nucleic acid molecule or the vector, and a in vitro method that comprises delivering the nucleic acid molecule or vector to an isolated mammalian cell to form a cell that expresses the fusion protein.

In more additional aspects, the present invention provides the fusion protein, the nucleic acid, the mammalian cell vector or wave of the present invention, for use in the treatment of a mammal. In one embodiment, the mammal has macular degeneration related to wet age or proliferative diabetic retinopathy. In another embodiment, the mammal has cancer. In another embodiment, the mammal has rheumatoid arthritis. In another embodiment, the mammal has asthma. In another embodiment, the mammal has osteoporosis.

Additional aspects and embodiments of the present invention are set forth in the appended claims.

Brief description of the drawings

Fig. 1. Flexible region of the 9-Gly linker in construction D2-9Gly-Fc. The relative flexibility predicted by the method of Karpus and Schultz (1985) shows the linker of 9 units of polyglycine (9-Gly) (amino acids 94 to 103) in the D2-9Gly-Fc protein as a region with greater flexibility than the average (> 1) compared to the D2-Fc construct that does not contain the 9-Gly linker. Both fusion proteins contain identical deamino acid sequences enclosed in the boxes: sp-signal peptide (amino acids -24 to -1), domain 2 of Flt-1 (amino acids 1 to 93) and the remains of IgG1-Fc (244 amino acids). The arrow represents the site of cleavage by the predicted signal peptide using the SignalP V2.0 program (Nielsen et al., 1997).

Fig. 2. Biological activity of D2-9Gly-Fc versus D2-Fc. 293 cells were cultured in the deprivation medium (M199 + 5% FCS) and transfected with plasmids containing the D2-9Gly-Fc and D2-Fc expression cassettes under the CMV promoter control. The conditioned medium (CM) was collected 72 h later. HUVEC were seeded in 96-well plates (2E3 cells / well) in deprivation medium + VEGF (10 ng / ml) and 50 µl of CM plus VEGF (10 ng / ml) was added 24 h later. Controls (+/- VEGF) were incubated with CM from the transfection of control plasmid pEGFP (Clontech; pEGFP carries a red-shifted variant of the green fluorescent green-type protein (GFP) that has been optimized for a brighter fluorescence and greater expression in mammalian cells). The positive control was treated with 50 ng of recombinant Flt-1-IgG protein (R&D Systems). The HUVEC were tested for proliferation 3 days after treatment using the CellTiter 96® AQueous reagent (Promega). The data represent the means of the OD490 average values of two experiments each tested in triplicate.

Fig. 3. Western blot analysis of D2-9Gly-Fc and D2-Fc. It seems that the size of both D2-9Gly-Fc and D2-Fc proteins is twice as large when it migrates in a non-reducing gel compared to the engel reducer migration. Proteins were loaded from the conditioned medium after transfections in 293 plasmid cells expressing D2-9Gly-Fc and D2-Fc were electrophoresed in SDS and transferred to a PVDF membrane. The transfer was probed with goat anti-Fc antibodies from human IgG1 and rabbit anti-goat IgG-HRP.

Fig. 4. Hybrid proteins sFlt-1 containing the 9Gly and VEGF Ex3 linker. Structure comparison of D29Gly-Ex3 / CH3 with previously constructed proteins. All three proteins contain the same amino acid sequence of domain 2 of Flt-1, which consists of 24 amino acids of the signal peptide of Flt-1 and 93 amino acids of domain 2 of Flt-1. D2-9Gly-Ex3 / CH3 contains 9 amino acids of the 9Gly linker, 14 amino acids of VEGF Ex3 and 120 amino acids of the CH3 region of human IgG1 heavy chain Fc.

Fig. 5. Biological activity of D2-9Gly-Ex3 / CH3 versus D2-9Gly-Fc. D2-9Gly-Ex3 / CH3 protein, in which domain 2 is connected to the CH3 region through the 9Gly linker and VEGF Ex3, also effectively inhibits VEGA-dependent HUVEC proliferation compared to D2- control proteins. 9Gly-Fc and D2-Fc. 50 ng of recombinant Flt-1-IgG (R&D Systems) was used as a control.

Fig. 6. HUVEC proliferation assay comparing the protein activity of D2- (Gly4Ser) 3-Fc with D2-9Gly-Fc and D2-9Gly-Ex3 / CH3.

Fig. 7. Western transfer. Proteins (not reduced and reduced) were separated from the conditioned medium of transfected 293 cells (15 µL CM) with plasmids expressing proteins (1): D2-9Gly-Fc; (2): D2- (G4S) 3-Fc and (3): EGFP by SDS electrophoresis and transferred to a PVDF membrane. The transfer was probed with goat anti-Fc antibodies from human IgG1 and goat anti-goat IgG-HRP.

Fig. 8. Combinations with / without linker 9Gly or VEGF Ex3. Structure comparison of three new proteins with or without 9Gly linker and / or VEGF Ex3, D2-9Gly-CH3, D2-CH3 and D2-Ex3 / CH3.

Fig. 9. HUVEC proliferation assay with the Flt-1 (D2) constructs with combinations 9Gly, Ex3 and CH3. The conditioned medium of 293 cells (5 µl) containing the proteins D2-Ex3 / CH3, D2-9Gly-CH3 and D2-CH3 were compared with D2-9Gly-Fc and D2-9Gly-Ex3 / CH3.

Fig. 10. Western transfer. 293 cells were transfected with plasmids expressing: (1) D2-9Gly-Fc; (2) D2-9Gly-CH3 (52/26 kDa); and (3) D2-CH3 (50/25 kDa). The 293 cell conditioned medium proteins (15 µl of non-reduced and / or reduced CM) were separated by electrophoresis in SDS and transferred to a PVDF membrane.The transfer was probed with HRP human anti-VEGF-R1 conjugate ( R&D Systems).

Fig. 11. VEGF "in vitro" binding assay. The conditioned media of 293 cells containing known concentrations of both D2-9Gly-Fc and soluble control receptors Flt-1 D (1-3) (varying in concentrations of 0.29-150 pM) were serially diluted ) and mixed with 10 pM VEGF. The amount of VEGF not bound by ELISA was then measured. D2-9 Gl-Fc joins VEGF with greater affinity than all other constructions. "n" represents the number of independent experiments (transfection and binding assay).

Fig. 12. The binding kinetics of soluble Flt-1 constructs were measured by surface plasmon resonance with a BIAcore instrument. The sFlt-1 constructs were immobilized on a detector chip, and VEGF165 was injected at concentrations ranging from 0.2 to 15 nM. The sensograms were evaluated using the BIAEvaluation program, the speed constants Ka and Kd were determined and the dissociation constant (KD) was calculated from the ratio Kd / Ka = KD. A lower KD value means better affinity.

Fig. 13A-13C. Fig. 13A shows the expression levels of the Flt-1 constructs that had various linkers. Fig. 13B shows the dimerization or multimerization of Flt-1 constructs having various linkers and a CH3 moiety of IgG1 Fc. The difference between the non-reduced and reduced conditions indicates that the proteins had multimerized. Fig. 13C shows the inhibitory bioactivity of the indicated Flt-1 constructs present in conditioned medium in a HUVEC proliferation assay in the presence of VEGF. Each of the constructs showed inhibitory activity that approaches the proliferation levels of the HUVEC in the absence of VEGF.

Fig. 14. Using a murine model of oxygen-induced retinopathy (OIR) of retinal neovascularization (NV), one of the Flt-1 constructs was administered to the mouse eyes and neovascularization was determined. The mice were exposed to hyper-toxic conditions. The amount of neovascular events was determined in the treated eyes compared to the events in the untreated eyes of the same animals. The animal was considered a "responder" if there was a reduction of more than 50% in neovascular events.

Detailed description of the invention

It is a discovery of the present invention that the Ig 2 Flt-1 type domain without domains 1 and 3 is capable of efficiently binding to VEGF and inhibiting the proliferation of VEGF-dependent endothelial cells. Domain 2 can be covalently linked to a multimerization domain through a linker. The linkers are typically polypeptide chains. The chain length may be 6, 7, 9, 11, 13, 15 or more amino acid residues, but typically it is between 5 and 25 residues. Depending on the length and composition of the side chain, a linker may have, but does not need, greater than average flexibility. Flexibility can be calculated using algorithms known in the art. Multimerization domains are those parts of multimeric proteins that promote the association of subunits for form, for example, dimers, trimers, tetramers, etc. Recombinant proteins suitable for effective binding to VEGF and / or inhibition of VEGF-dependent endothelial cell proliferation are selected from the group consisting of SEQ ID Nos. 2, 8, 21, 23, and 25.

In addition, it has been discovered that multimerization domains and linkers can be used with a variety of different proteins or parts of proteins to induce multimerization. Such proteins may be those that bind a ligand or receptor only when they are multimerized, or they may be those whose binding affinity is enhanced when they are multimerized. Suitable multimerization proteins include extracellular receptors (including parts thereof), variable regions of antibodies, cytokines, chemokines, and growth factors. Suitable proteins include receptor tyrosine kinase and receptor serine-threonine kinase. Specific examples of extracellular receptors include the EGF receptor, G-protein coupled receptors, the FGF receptor, Fc receptors, T cell receptors, etc. Examples of variable regions of antibodies include Fab, F (ab ') 2, and ScFv. Examples of cytokines include GM-CSF, IL1a, IL-11, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12 , IL-18, IL-21, IL-23, IFN-a, IFN-1, IFN-y, MIP-1a, MIP-11, TGF-1, TNFa, and TNF1. Examples of chemokines include BCA-1 / BLC, BRAK, chemokine CC-2, CTACK, CXCL-16, ELC, ENA, ENA-70, ENA-74, ENA-78, eotaxin, exodus-2, fractalkine, GCP- 2, GRO, GRO alpha (MGSA), GRO-beta, GRO-gamma, HCC-1, HCC-4, I-309, IP-10, I-TAC, LAG-1, LD78-beta, LEC / NCC- 4, LL-37, lymphotactin, MCP, MCAF (MCP-1), MCP-2, MCP-3, MCP-4, MDC, MDC, MDC-2, MDC-4, MEC / CCL28, MIG, MIP, MTP -1 alpha, MIP-1 beta, MIP-1 delta, MIP-3 / MPIF-1, MIP-3 alpha, MIP-3 beta, MIP-4 (PARC), MIP-5, NAP-2, PARC, PF -4, RANTES, RANTES-2, SDF-1 alpha, SDF-1 beta, TARC, and TECK. Examples of growth factors include human anfirregulin, human angiogenesis proteins, human ACE, human angiogenin, human angiopoietin, human angiostatin, human betacellulin, human BMP, human BMP-13 / CDMP-2, human BMP-14 / CDMP-1, Human BMP-2, human BMP-3, human BMP-4, human BMP-5, human BMP-6, human BMP-7, human BMP-8, human BMP-9, human colony stimulating factors, human flt3 ligand, G -CSFhuman, human GM-CSF, human M-CSF, human connective tissue growth factor, human crypto-1, human cryptic, human ECGF, human EGF, human EG-VEGF, human erythropoietin, human fetuin, human FGF, FGF -1 human, FGF-10 human, FGF-16 human, FGF-17 human, FGF-18 human, FGF-19 human, FGF-2 human, FGF-20 human, FGF-3 human, FGF-4 human, FGF -5 human, human FGF-6, human FGF-7 / KGF, human FGF-8, human FGF-9, human FGF-basic, human FGF-basic, human GDF-11, human GDF-15, releasing factor human growth hormone, HB-E Human GF, human heregulin, human HGF, human IGF, human IGF-I, human IGF-II, human inhibit, human KGF, human LCGF, human LIF, miscellaneous human growth factors, human MSP, human myostatin, human myostatin propeptide , human nerve growth factor, human oncostatin M, human PD-ECGF, human PDGF, human PDGF (AA homodimer), human PDGF (AB heterodimer), human PDGF (BB homodimer), human PDGF (CC homodimer), human PIGF, Human PIGF, human PIGF-1, human PIGF-2, human SCF, human SMDF, human stem cell growth factors, human SCGF-alpha, human SCGF-beta, human thrombopoietin, human transforming growth factor, human TGF-alpha, Human TGF-beta, and human VEGF.

The Flt-1 receptor protein has an extracellular part comprising seven Ig-like domains. These are located in the remains with the numbers 32 ... 123, 151 ... 214, 230 ... 327, 335 ... 421, 428 ... 553, 556 ... 654, and 661 ... 747 of Genbank accession number P17948, see also SEQ ID No. 15. The remains with numbers 1-26 comprise a signal sequence. The Flt-1 protein is encoded by the DNA sequence shown in the Genbank accession number NM_002019 (SEQ ID NO: 14).

The multimerization domains that can be used in accordance with the present description are known in Latin America. The sequences of the Fc part of the heavy chain of IgG1 or gamma IgG2 can be used, for example, CH3 alone (amino acids 371-477) or domains both CH2 and CH3 (amino acids 247-477). The Fc part of the Ig molecules is that obtained by cleavage of complete antibody molecules with the enzyme papain. Other means may be used to obtain these parts. For the heavy chain protein sequence of IgG1 gamma, see Genbank accession number Y14737 and SEQ ID NO. 10. Other Fc regions may be used, for example, other types of IgG and IgA, IgM, IgD antibodies, or IgE. The VEGF multimerization region can also be used. A DNA sequence encoding VEGF in Genbank accession number NM_003376 and ECS ID No. 11 is shown. A VEGF amino acid sequence is shown in Genbank accession number CAC19513 and SEQ ID No. 12. The multimerization region of VEGF (SEQ ID No. 13), encoded by exon 3 of VEGF (VEGF Ex3), is at approximately amino acid residues 75-88 of the VEGF protein (SEQ ID No. 12). Multimerization domains will cause at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 75%, 80%, 85%, 90%, or 95% of monomeric fusion proteins look on a non-denaturing polyacrylamide gel at a speed suitable for a multimer. Glycosylation can affect the migration of a protein in a gel. Although particular sequences are shown here, variants such as allelic variants can also be used. Typically, said variants will have at least 85%, 90%, 95%, 97%, 98%, or 99% identity with the sequence described.

Multimerization can be tested, for example, using reducing and non-reducing gels, as demonstrated in this document. Multimerization can also be assayed by detection of increased binding affinity of a protein by its ligand / receptor. BiaCore ™ surface plasmon resonance assays can be used in this regard. These tests detect changes in mass by measuring changes in the index of refraction in an aqueous layer near the surface of a detector chip. Any method known in the art can be used to detect multimerization.

The linker moieties according to the present description may be composed of, for example, 5-100 amino acid residues, 5-75 amino acid residues, 5-50 amino acid residues, 5-25 amino acid residues, 5-20 amino acid residues, 5-15 residues amino acids, 5-10 amino acid residues, or 5-9 amino acid residues. Examples of useful linkers include: gly9 (SEQ ID No. 27), glu9 (SEQ ID No. 28), ser9 (SEQ ID No. 29), gly5cyspro2cys (SEQ ID No. 30), (gly4ser) 3 (SEQ ID No. 31), SerCysValProLeuMetArgCysGlyGlyCysCysAsn (SEQ ID No. 32), ProSerCysValProLeuMetArgCysGlyGlyCysCysAsn (SEQ ID No. 13), GlyAspLeuIleTyrArgAsnGlnLys (SEQ ID No. 26), and Gly9ProSerCysValProLeuMetArgCysGlyn (IDC No. 34). Other polypeptide linkers that can be used include a polyglycine of different lengths, including 5, 7, or 30 moieties. In addition, other parts of Flt-1 can be used as linker, for example, domain 3 of Flt-1. See SEQ ID No. 15. Linker moieties can also be created from other polymers, such as polyethylene glycol. Such linkers may have 10 to 1000, 10-500, 10-250, 10-100, or 10-50 monomer units of ethylene glycol. The polyrosadecuados should be of a size similar to the size occupied by the appropriate range of amino acid residues. A typical sized polymer would provide a spacing of approximately 10-25 angstrom.

Despite the generality of the foregoing description, however, the present invention relates to a defusion protein of formula XYZ, wherein: X comprises a portion of the VEGF receptor, Y is a linker moiety and Z is a multimerization domain , wherein the linker moiety Y is a polypeptide of 5-50 amino acid residues that has an above-average flexibility as determined by the method of predicting flexibility of Karpus & Schultz, 1985, Naturwiss, 72: 212-213, and wherein said fusion protein binds to VEGF when multimerized.

Fusion proteins according to the present description, including the fusion proteins of the present invention, can be created by any means known in the art. Although such proteins can be created by synthetic deformation, or by joining parts that are made, recombinant production can also be used. A fused gene sequence can be produced using conventional recombinant DNA tools. The fused gene sequence can be inserted into a vector, for example, a viral or plasmid vector, to replicate the fused gene sequence. A promoter sequence that is functional in the final receptor cell can be introduced upstream of the promoter gene sequence. The promoters used can be constitutive, inducible or suppressable. Examples of each type are well known in the art. The vector may be introduced into a mammalian or host cell by any means known in the art. Suitable vectors that can be used include adenovirus, adeno-associated virus, retrovirus, lentivirus, and plasmids. If the vector is in a viral vector and the vector has been packaged, then virions can be used to infect cells. If naked DNA is used, then the transfection or transformation procedures that are appropriate for the particular cell choppers can be used. Nude DNA formulations can be used using polymers, liposomes, or nanospheres for the delivery of fusion genes. Cells that can be transformed or transfected with recombinant constructs according to the invention can be any that are suitable for the specialist. Exemplary cell types that can be used include bacteria, yeasts, insect, and mammalian cells. Among mammalian cells, cells of many tissue types may be chosen, as appropriate. Exemplary cells that can be used are fibroblasts, hepatocytes, endothelial cells, stem cells, hematopoietic cells, epithelial cells, myocytes, neuronal cells, and keratinocytes. These cells can be used to produce proteins in vitro, or they can be supplied to mammals including humans to produce the encoded proteins in vivo. This means of delivery is an alternative to the supply of nucleic acid to a mammal, to the supply of a viral vector to a mammal, and to the supply of the fusion protein to a mammal.

The protein or nucleic acid compositions may be in vehicles, such as buffers, aqueous or lipophilic, sterile or non-sterile, pyrogenic or non-pyrogenic vehicles. Non-pyrogenic vehicles are useful for injectable formulations. The formulations can be liquid or solid, for example, lyophilized. The formulations can also be administered in the form of aerosols. The compositions may contain one or more fusion proteins or one or more nucleic acids, or both fusion proteins and nucleic acids. The fusion proteins and / or nucleic acids in a composition may be homogeneous, in which case homomultimeric proteins will be formed, or they may be heterogeneous in the composition, in which case heteromultimeric proteins will be formed. In the case of heteromultimers, typically the remainder X will vary between fusion proteins, but the remainder Z will be the same among the fusion proteins.

Fusion proteins can be provided to a mammalian cell or host by any means known in Latin America. The protein can be supplied to the cell or host. The nucleic acid can be administered to the host cell. Transformed or transfected cells can be administered to the cell or host. In the latter case, cells of the same genetic background are desired to reduce transplant rejection.

Suitable cells for delivery to mammalian host animals include any mammalian cell type of any organ, tumor, or cell line. For example, human, murine, zebra, sheep, bovine, dog, cat, and pig cells can be used. Cell types suitable for use include, without limitation, fibroblasts, hepatocytes, endothelial cells, keratinocytes, hematopoietic cells, epithelial cells, myocytes, neuronal cells, and stem cells.

Means of supply of fusion proteins or nucleic acids encoding fusion proteins include the supply of cells expressing fusion proteins, the supply of fusion proteins, and the supply of nucleic acids encoding fusion proteins. Fusion proteins, cells, or nucleic acids can be delivered directly to the desired organ or tumor, for example, by injection, catheterization, or endoscopy. They can also be given intravenously, intrabronchially, intratumorally, intrathecally, intramuscularly, intraocularly, topically, subcutaneously, transdermally or per os. Patients that can be treated effectively include those with wet age-related macular degeneration, proliferative diabetic retinopathy, rheumatoid arthritis, osteoarthritis, uveitis, asthma, and cancer. The treatments will improve the symptoms and / or markers of the disease and / or the severity of the disease.

Nucleic acids can be supplied to mammals, and in particular to humans, in any desired vector. These include viral or non-viral vectors, including adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, and plasmid vectors. Exemplary types of viruses include HSV (herpes simplex virus), adenovirus, AAV (adeno-associated virus), HIV (human immunodeficiency virus), VIB (bovine immunodeficiency virus), and VLM (murine leukemia virus) . Nucleic acids can be administered in any desired format that provides sufficiently effective delivery levels, including in viral particles, in liposomes, in nanoparticles, and in complex with polymers.

Combinations of protein and nucleic acid treatments can be used. For example, a fusion protein according to the present description can be administered, for example, a fusion protein according to the present invention to a patient. If a favorable response is observed, then a nucleic acid molecule encoding the fusion protein can be administered for a long-term effect. Alternatively, the protein and nucleic acid can be administered simultaneously or approximately simultaneously. In another alternative, an antibody or fusion protein can be administered for a ligand followed by or concomitantly with an antibody or fusion partner for a receptor. Another option employs a combination of nucleic acids in which one encodes an antibody and others encodes a fusion protein. Some antibodies that can be used in combination with the Flt-1 constructs of the present disclosure, for example, combination with the Flt-1 constructs of the present invention (either in the form of protein or nucleic acid) are bevacizumab and ranibizumab, both directed against VEGF. These are particularly useful for treating cancer and macular degeneration, respectively.

The practice of the present description, including the practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which belong to the abilities of The technique. These techniques are fully explained in the literature, such as, Molecular Cloning: A Laboratory Manual, Second Edition (Sambrook et al., 1989); Current Protocols In Molecular Biology (F.M. Ausubel et al., Eds., 1987); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Animal Cell Culture (R.I. Freshney, ed., 1987); Methods In Enzymology (Academic Press, Inc.); Handbook Of Experimental Immunology (D.M. Wei & C.C. Blackwell, eds.); Gene Transfer Vectors For Mammalian Cells (J.M. Miller & M.P. Calos, eds., 1987); PCR: The Polymerase Chain Reaction (Mullis et al., Eds., 1994); Current Protocols In Immunology (J.E. Coligan et al, eds., 1991); Antibodies: A Laboratory Manual (E. Harlow and D. Lane eds. (1988)); and PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. (1995)).

A gene delivery vehicle is any molecule that can transport polynucleotides inserted into a host cell. Examples of gene delivery vehicles are liposomes, biocompatible polymers, including natural polymers and synthetic polymers; lipoproteins; polypeptides; polysaccharides; lipopolysaccharides; artificial viral wraps; metal particles; and bacteria, viruses, such as baculovirus, adenovirus and retrovirus, bacteriophage, cosmid, plasmid, fungal and other recombination vehicles typically used in the art that have been described for expression in a variety of eukaryotic and prokaryotic hosts, and can be used for therapy gene as well as for the simple expression of proteins.

Gene delivery, gene transfer, and the like, as used herein, are terms that refer to the introduction of an exogenous polynucleotide (sometimes referred to as "transgene") into a host cell, regardless of the method used for introduction. Such methods include a variety of well-known techniques such as vector-mediated gene transfer (for example, viral infection / transfection, or various different protein-based or different lipid-based gene delivery complexes) as well as techniques that facilitate polynucleotide delivery. "nudes" (such as electroporation, "gene gun" delivery and various different techniques used for the introduction of polynucleotides). The introduced polynucleotide can be maintained stably or transiently in the host cell. Stable maintenance typically requires that the introduced polynucleotide contain a replication origin compatible with the host cell or be integrated into a replicon of the host cell such as an extrachromosomal replicon (eg, a plasmid) or a nuclear or mitochondrial chromosome. It is known that several vectors are capable of mediating the transfer of genes to mammalian cells, which are known in the art and are described herein.

The exogenous polynucleotide is inserted into a vector such as adenovirus, partially removed adenovirus, completely removed adenovirus, adeno-associated virus (AAV), retrovirus, lentivirus, naked plasmid, plasmid / liposome complex, etc. for delivery to the host intravenously, intramuscularly, through the portal or other route of administration. Expression vectors that can be used in the methods and compositions of the present description, for example, in the compositions of the present invention include, for example, viral vectors. One of the most frequently used methods of gene therapy administration, both in vivo and ex vivo, is the use of viral vectors for gene delivery. Many devirus species are known, and many have been studied for gene therapy purposes. The most commonly used viral vectors include those derived from adenovirus, adeno-associated virus (AAV) and retroviruses, including lentiviruses, such as human immunodeficiency virus (HIV).

The adenovirus is a nuclear DNA virus, enveloped with a genome of approximately 36 kb, which has been well characterized through studies in classical genetics and molecular biology (Hurwitz, MS, AdenovirusesVirology, 3rd edition, Fields et al., Eds. , Raven Press, New York, 1996; Hitt, MM et al., Adenovirus Vectors, The Development of Human Gene Therapy, Friedman, T. ed., Cold Spring Harbor Laboratory Press, New York 1999).

Viral genes are classified into early (called E1-E4) and late (called L1-L5) transcriptional units, which refer to the generation of two temporary classes of viral proteins. The demarcation of these events is the replication of viral DNA. Human adenoviruses are divided into numerous serotypes (approximately 47, numbered accordingly and classified into 6 groups: A, B, C, D, E and F), based on properties that include hemagglutination of red blood cells, oncogenicity, compositions and homology of DNA and protein amino acids, and antigenic relationships.

Recombinant adenoviral vectors have several advantages for use as degenes delivery vehicles, including tropism by both dividing and non-dividing cells, minimal pathogenic potential, ability to replicate at a high titer for the preparation of vector stores, and potential to carry large inserts (Berkner, KL, Curr. Top. Micro. Immunol. 158: 39-66, 1992; Jolly, D., Cancer Gene Therapy 1: 5164 1994). Adenoviral vectors have been designed with deletions of various adenoviral gene sequences, such as partially eliminated pseudoadenoviral (PAV) and adenoviral (called "DeAd") vectors, to exploit the desirable characteristics of adenoviruses that make them a suitable vehicle for nucleic acid delivery. to recipient cells.

In particular, pseudo-adenoviral (PAV) vectors, also known as 'gutless adenovirus' or mini-adenoviral vectors, are adenoviral vectors derived from the genome of an adenovirus that contains the minimum cis-acting nucleotide sequences necessary for replication and packaging of the vector genome and They may contain one or more transgenes (See, U.S. Patent No. 5,882,877 covering the pseudodenovirus vectors (PAV) and methods for producing PAV). PAVs have been designed to take advantage of the desirable characteristics of adenoviruses that make them a suitable vehicle for the supply of genes. Although adenoviral vectors can generally carry inserts up to 8 kb in size due to the deletion of regions that are dispensable for viral growth, maximum loading capacity can be achieved with the use of adenoviral vectors containing deletions of most viral coding sequences, including PAV See, U.S. Patent No. 5,882,877 to Gregory et al .; Kochanek et al., Proc. Natl Acad. Sci. USA 93: 5731-5736, 1996; Parks et al., Proc. Natl Acad. Sci. USA 93: 13565-13570, 1996; Lieber et al., J. Virol. 70: 8944-8960, 1996; Fisher et al., Virology 217: 11-22, 1996; United States Patent No. 5,670,488; PCT Publication No. WO96 / 33280, published October 24, 1996; PCTN Publication WO96 / 40955, published on December 19, 1996; PCT Publication No. WO97 / 25446, published July 19, 1997; PCT Publication No. WO95 / 29993, published November 9, 1995; PCT Publication No. WO97 / 00326, published January 3, 1997; Morral et al., Hum. Gene Ther. 10: 2709-2716,1998. Such PAVs, which can accommodate up to approximately 36 kb of foreign nucleic acids, are advantageous because the vector's loading capacity is optimized, while reducing the potency of host immune responses against the vector or the generation of competent replication viruses. PAV vectors contain the 5 'inverted terminal repeat (ITR) and 3' ITR denucleotide sequences that contain the origin of replication, and the cis-acting nucleotide sequence necessary for the PAV genome packaging, and can accommodate one or more transgenes with appropriate regulatory elements, for example, promoter, enhancers, etc.

Others, partially deleted adenoviral vectors provide a partially deleted adenoviral vector (called "DeAd") in which most of the early adenoviral genes necessary for virus replication are removed from the vector and placed within the chromosome of a producing cell under the control of a conditional promoter. The adenoviral genes that can be removed that can be placed in the producer cell can include E1A / E1B, E2, E4 (only ORF6 and ORF6 / 7 have to be placed in the cell), pIX and pIVa2. E3 can also be removed from the vector, but since it is not necessary for the production of the vector, it can be omitted from the producing cell. Late adenoviral genes, usually under the control of the main late promoter (MLP), are present in the vector, but the MLP can be replaced by a conditional promoter.

Conditional promoters suitable for use in DeAd vectors and producer cell lines include those with the following characteristics: low basal expression in an uninduced state, so that no cytotoxic or cytostatic adenoviral genes are expressed at levels harmful to the cell; and expression of high level induced state, so that sufficient amounts of viral proteins are produced to support replication and assembly of the vector. Preferred conditional promoters suitable for use in DeAd vectors and producer cell lines include the dimerizer gene control system, based on the FK506 and rapamycin immunosuppressive agents, the ecdysone gene control system and the tetracycline gene control system. It may also be useful in the present description, including the present invention, the GeneSwitch ™ technology (Valentis, Inc., Woodlands, TX) described in Abruzzese et al., Hum. Gene Ther. 1999 10: 1499-507. The partially removed adenoviral expression system is further described in WO99 / 57296.

The adeno-associated virus (AAV) is a single-stranded human DNA parvovirus whose genome is 4.6 kb in size. The AAV genome contains two main genes: the rep gene, which encodes the rep proteins (Rep 78, Rep68, Rep 52, and Rep 40) and that are involved in AAV replication, rescue, transcription and integration; and the gencap that encodes the cap proteins that form the AAV viral particle. The AAV obtains its dependence on a denovirus or other auxiliary virus (for example, herpesvirus) to deliver the essential gene products that allow the AAV to experience a productive infection, that is, reproduce itself in the host cell. In the absence of the auxiliary virus, AAV is integrated as a provirus into the chromosome of the host cell, until it is rescued by superinfection of the host cell with an auxiliary virus, usually an adenovirus (Muzyczka, Curr. Top. Micor. Immunol. 158: 97-127, 1992).

The interest in AAV as a gene transfer vector is caused by several characteristics of its biology. In both cases of the AAV genome there is a nucleotide sequence known as inverted terminal repeat (ITR), which contains the cis-acting nucleotide sequences necessary for virus replication, rescue, packaging and integration. The integration function of the ITR mediated by the rep protein in trans allows the AAV genome to integrate into a cell chromosome after infection, in the absence of virus-assist. This unique property of the virus is relevant for the use of AAV in gene transfer, since it allows the integration of a recombinant AAV that contains a gene of interest in the cellular genome. Therefore, stable genetic transformation can be achieved, ideal for many of the objectives of degenes transfer, through the use of rAAV vectors. In addition, the integration site for AAV is well established and has been located on chromosome 19 of the human being (Kotin et al., Proc. Natl. Acad. Sci. 87: 2211-2215, 1990). This predictability of the integration site reduces the danger of random insertion events in the genomacellular that can activate or inactivate host genes or interrupt the coding sequences, consequences that can limit the use of vectors with AAV integration, the elimination of this gene in the design of rAAV vectors can cause altered integration patterns that have been observed with rAAV vectors (Ponnazhagan et al., Hum Gene Ther. 8: 275-284, 1997).

There are other advantages to using AAV for gene transfer. The host range of AAVs is wide, and unlike retroviruses, AAVs can infect both quiescent and dividing cells. In addition, AAVs have not been associated with human disease, ignoring many of the concerns that have arisen with gene transfer vectors derived from retroviruses.

Conventional approaches to the generation of recombinant rAAV vectors have required the coordination of a series of intracellular events: the transfection of the host cell with a rAAV vector genome containing a transgene of interest flanked by the AAV ITR sequences, the transfection of the host cell by a plasmid that encodes genes for the AAV rep and cap proteins that are necessary in trans, and infection of the cell transfected with an auxiliary virus to deliver the non-AAV auxiliary functions necessary in trans (Muzyczka, N., Curr Top, Micor. Immunol. 158: 97-129, 1992). Adenoviral proteins (or other auxiliary viruses) activate transcription of the AAV rep gene, and rep proteins can then activate the transcription of AAV cap genes. The cap proteins then use the ITR sequences to package the rAAV genome into a rAAV viral particle. Therefore, the effectiveness of the packaging is determined, in part, by the availability of adequate amounts of the structural proteins, as well as the accessibility of any necessary cis-acting packaging sequence in the rAAV vector genome.

Retroviral vectors are a common tool for gene delivery (Miller, Nature (1992) 357: 455460). The ability of retroviral vectors to provide a single unordered copy of the gene in a wide range of somatic cells from rodents, primates and humans makes retroviral vectors very suitable for transferring genes to a cell.

Retroviruses are RNA viruses in which the viral genome is RNA. When a host cell is infected with unretrovirus, genomic RNA is reverse transcribed into a DNA intermediate that integrates very effectively into the chromosomal DNA of infected cells. This integrated DNA intermediate is known as comoprovirus. The transcription of the provirus and its assembly into infectious viruses occurs in the presence of an appropriate auxiliary virus or in a cell line containing appropriate sequences that allow encapsidation without simultaneous production of a contaminating auxiliary virus. An auxiliary virus is not necessary for the production of recombinant retrovirus if the sequences are provided for encapsidation by co-transfection with appropriate vectors.

The retroviral genome and pro viral DNA have three genes: gag, pol, and env, which are flanked by two long terminal repeat sequences (LTR). The gag gene encodes internal structural proteins (matrix, capsid, and nucleocapsid); the pol gene encodes RNA-directed DNA polymerase (reverse transcriptase) and the gene encodes the viral envelope glycoproteins. The 5 'and 3' LTRs serve to promote transcription and lapolydenylation of virion RNAs. The LTR contains all other cis action sequences necessary for viral replication. Lentiviruses have additional genes that include vit, vpr, tat, rev, vpu, nef, and vpx (in HIV1, HIV-2 and / or VIS). Adjacent to the LTR 5 'there are sequences necessary for the reverse transcription of the genome ( the binding site of the tRNA primer) and for the effective encapsidation of the viral RNA in particles (the Psi site). If the sequences necessary for encapsidation (or packaging of retroviral RNA in infectious virions) are absent in the viral genome, the result is a defect in cis that prevents encapsidation of genomic RNA. However, the resulting mutant is still capable of directing the synthesis of all virion proteins.

Lentiviruses are complex retroviruses that, in addition to the common retroviral genes gag, pol and env, contain other genes with regulatory or structural function. The greater complexity makes it possible for lentiviruses to modulate their life cycle, as in the course of latent infection. A typical lentivirus is the human immunodeficiency virus (HIV), the etiologic agent of AIDS. In vivo, HIV can infect terminal differentiated cells that rarely divide, such as lymphocytes and macrophages. In vitro, HIV can infect primary cultures of monocyte-derived macrophages (MDM) as well as HeLa-Cd4 or T-lymphoid cells stopped in the cell cycle by treatment with afidicoline or gamma irradiation. Infection of cells depends on the active contribution of HIV preintegration complexes through the nuclear pores of the target cells. This is caused by the interaction of multiple partially redundant molecular determinants in the complex with the nuclear import machinery of the target cell. The determinants identified include a functional nuclear localization signal (NLS) in the gag matrix protein (MA), the cariophilic virion-associated protein, vpr, and a C-terminal phosphotyrosine residue in the MA gag protein. The use of retroviruses for gene therapy is described, for example, in US Patent 6,013,516; and U.S. Patent 5,994,136.

Other methods to deliver DNA to cells do not use viruses for delivery. For example, cationic amphiphilic compounds can be used to deliver the nucleic acid of the present disclosure, for example, the acidic acid of the present invention. As the compounds designed to facilitate intracellular delivery of biologically active molecules must interact with both non-polar and polar environments (in or on, for example, the plasma membrane, tissue fluids, compartments within the cell, and the biologically active molecule itself), said compounds They are typically designed to contain both polar and polar polar domains. The compounds having these two domains can be called amphiphiles, and many synthetic lipids and lipids that have been described for use in facilitating said intracellular delivery (whether for in vitro or in vivo application) meet this definition. A particularly important class of such amphiphiles is amphiphilophosionic. In general, cationic amphiphiles have polar groups that are capable of positively loading at or about physiological pH, and this property is understood to be important in the art to define how amphiphiles interact with the many types of biologically active (therapeutic) molecules including , for example, negatively charged polynucleotides such as DNA.

The use of compositions comprising cationic amphiphilic compounds for gene delivery is described, for example, in US Patent 5,049,386; US 5,279,833; US 5,650,096, US 5,747,471; US 5,767,099; US 5,910,487; US 5,719,131; US 5,840,710; US 5,783,565; US 5,925,628; US 5,912,239; US 5,942,634; US 5,948,925; US 6,022,874; the U.S. document 5,994,317; the U.S. document 5,861,397; the U.S. document 5,952,916; the U.S. document 5,948,767; The document

U.S. 5,939,401; and the U.S. document 5,935,936.

In addition, the nucleic acid of the present description, for example, the nucleic acid of the present invention can be supplied using "naked DNA". Methods for delivering a non-integrating, non-infectious DNA sequence encoding a desired polypeptide or peptide functionally linked to a promoter, free from association with proteins that facilitate transfection, viral particles, liposomal formulations, charged lipids and calcium phosphate precipitation agents U.S. Patent 5,580,859 is described; U.S. 5,963,622; the U.S. document 5,910,488.

Gene transfer systems that combine viral and non-viral components have also been reported. Cristiano et al., (1993) Proc. Natl Acad. Sci. USA 90: 11548; Wu et al. (1994) J. Biol. Chem. 269: 11542; Wagner et al. (1992) Proc. Natl Acad. Sci. USA 89: 6099; Yoshimura et al. (1993) J. Biol. Chem. 268: 2300; Curiel et al. (1991) Proc. Natl Acad. Sci. USA 88: 8850; Kupfer et al. (1994) Human Gene Ther. 5: 1437; and Gottschalk et al. (1994) Gene Ther. 1: 185. In most cases, adenoviruses have been incorporated into gene delivery systems to take advantage of their endosomolytic properties. The presented combinations of viral and non-viral components generally involve covalent binding of the adenovirus to a gene delivery complex or co-adenovirization of unbound adenovirus with cationic lipid complexes: DNA.

For the supply of DNA and protein to the eye, administration will typically be local. This has the advantage of limiting the amount of DNA that you have to administer and limiting the systemic side effects. Many possible modes of delivery may be used including, but not limited to: topical administration on the cornea by a gene gun; subconjunctival injection, intracameral injection, by eye drops to the cornea, interior injection of the anterior chamber by temporal limbus, intrastromal injection, application to the cornea combined with electrical pulses, intracorneal injection, subretinal injection, intravitreal injection, and intraocular injection. Alternatively, ex vivo cells can be transfected or transduced and delivered by intraocular implant. See, Auricchio, Mol. Ther. 6: 490-494, 2002; Bennett, Nature Med. 2: 649-654, 1996; Borrás, Experimental Eye Research 76: 643-652, 2003; Chaum, Survey of Ophtalmology 47: 449-469, 2002; Campochiaro, Expert Opinions in Biological Therapy 2: 537-544 (2002); Lai, Gene Therapy 9: 804 813, 2002; Pleyer, Progress in Retinal and Eye Research, 22: 277-293, 2003.

The effects of various therapeutic agents and administrations proposed in animal models suitable for particular diseases can be tested. For example, retinopathy of prematurity can be tested in a model of oxygen-induced retinopathy in mice as described in Smith, Investigative Ophtalmology & Visual Science, 35: 101-111, 1994. Laser-induced choroidal neovascularization can be used in mice as neovascularization models. human choroidal (CNV) that occurs in diseases such as age-related macular degeneration. Tobe, American Journal of Pathology 153: 1641-1646, 1998. Other CNV models have been developed in primates, rats, dwarf pigs, and rabbits. Mouse models of age-related macular degeneration have been developed in genetically deficient mice. Mice deficient in monocyte chemoattractant-1 protein or C-C chemokine receptor-2 develop age-related macular degeneration characteristics. Ambati, Nature Med. 9: 1390-1397,2003.

Although the invention has been described with respect to specific examples including currently preferred ways of carrying out the invention, those skilled in the art will appreciate that there are numerous variations and permutations of the systems and techniques described above that are within the scope of the appended claims.

Examples

Example 1

Two constructions were generated: the first, D2-9Gly-Fc, which contained a 9-unit polyglycine linker (9Gly) and the second, D2-Fc, with the same sequence except the 9Gly linker (Fig. 1).

The amino acid sequences of the D2-9Gly-Fc and D2-Fc proteins were analyzed using the Protein AnalysisToolbox of the Mac Vector 6.5.1 sequence analysis program. (IBI, New Haven, CT). The 9-unit polyglycine linker in the D2-9Gly-Fc sequence was identified as a region with greater average flexibility by the flexibility prediction method of Karpus and Schultz (1985) Naturwiss, 72: 212-213. This region was not detected in the sequence of D2-Fc (Fig. 1).

Example 2

An Ig 2 Flt-1 type domain connected to the Fc region of IgG1 was tested by a 9-unit linker of flexible polyglycine (D2-9Gly-Fc). The D2-9Gly-Fc fusion protein is capable of efficiently binding to VEGF and inhibiting the proliferation of human umbilical vein endothelial cells (HUVEC) dependent on VEGF. See Fig. 2. In contrast, when the Ig 2 Flt-1 type domain is directly linked to the IgG1 heavy chain (Fc) to form D2-Fc, only minimal binding to VEGF was observed. See Fig. 2. Both dimerization by IgG1 Fc and the insertion of a flexible linker appear to facilitate VEGF binding to domain 2 of Flt-1. The presence of dimeric forms was confirmed in both D2-9Gly-Fc and D2-Fc by Western blot analysis. See Fig. 3.

Example 3

An intravitreal injection of AAV vector (1 x 108 to 1 x 109 particles in a volume of 0.0005 ml) is administered to newborn C57BL / 6 (P0) or 1 day old (P1) mice. Retinal neovascularization (NV) is induced in C57BL / 6 mice exposing P7 pups and their lactation females to hyperoxia for 5 days. The offspring were returned to ambient air at P12 and sacrificed at P17 (time of NV peak). (Smith LEH, Weslowski E, McLellan A, Kostyk SK, D'Amato R, Sullivan and D'Amore PA. Oxygen-Induced Retinopathy in the Mouse. Invest Opth Vis Sci. 1994; 35: 101-111.) They were sectioned from serial cross-section complete eyes impregnated in paraffin at 5 micrometer intervals. The degree of NV is determined by counting the number of nuclei of internal endothelial cells to the internal limitation membrane in sections taken every 100 micrometers.

Cohorts of animals treated with AAV vectors encoding concohort anti-angiogenic agents treated with vectors encoding irrelevant transgenes or with vectors that do not encode a transgene are compared. The average number of endothelial cell nuclei in each treated eye is compared with Similar eye noted for each animal.

Example 4

Generation of D2-9Gly-Ex3 / CH3

It has been shown that domain 2 of Flt-1 is essential for VEGF165 binding. However, it was shown that domain 2 of Flt-1 was only unable to join VEGF A. (Davis-Smyth et al., 1996.) VEGF A, when present in dimer form, binds to Flt-1 through of acidic moieties (amino acids 63-67 of the mature protein) that allow a possible mechanism for receptor ligand-induced dimerization (Keyt et al, 1996).

Therefore, a dimerization of domain 2 of Flt-1 was used as a strategy to restore binding of domain 2 of Flt-1 to VEGF A. Fusion with an IgG heavy chain fragment can be used for dimerization of proteins (Davis -Smyth et al., 1996). Here it is shown that amino acids 75-88 (ie, PSCVPLMRCGGCCN; SEQ ID NO. 13) of VEGF A (SEQ ID NO. 12) increase the biological activity of the sFlt-1 hybrid proteins.

Initially, three hybrid proteins were designed: D2-9Gly-Fc, D2-Fc and D2-9Gly-Ex3 / CH3 (Fig. 4). The three hybrid proteins contain the same D2 domain of Flt-1 as D2-9Gly-Fc. No binding of VEGF with D2-Fc was observed, which does not contain the linker of 9 polyglycine units (9Gly). The third protein, D2-9Gly-Ex3 / CH3, contains the 9-unit polyglycine linker (9Gly) and the VEGF multimerization domain (amino acids PSCVPLMRCGGCCN; SEQ ID No. 13; VEGF Ex3), but also contains the CH3 region of Fc heavy chain of human IgG1 (amino acids 371-477 of SEQ ID NO: 10).

The D2-Fc protein showed no effective inhibitory activity in the HUVEC proliferation assay (Fig. 5) and by implication it did not bind VEGF165 effectively. However, the third hybrid protein, D2-9Gly-Ex3 / CH3, which comprises domain 2 of Flt-1 fused with the CH3 region through both the 9Gly linker and the VEGF165 (Ex3) dimerization region, showed inhibitory activity in one trial. VEGF-dependent HUVEC proliferation (Fig. 5). This implies that this hybrid protein binds to VEGF165 effectively.

Example 5

Use of the linker (Gly4Ser) 3 in the Flt-1 D2 construction

The use of several polyglycine linkers has been previously described for the improvement of protein characteristics (Mouz et al., 1996; Qiu et al., 1998). For the following construction another type of linker has been used, the oligomer of 15 units (Gly-Gly-Gly-Gly-Ser) 3 (Huston et al., 1988). The D2- (Gly4Ser) 3-Fc protein was generated and contains domain 2 of Flt-1, linker (Gly4Ser) 3 and the Fc region of the human IgG1 heavy chain.

D2- (Gly4Ser) 3-Fc was further characterized in a HUVEC proliferation assay. The biological activity of D2- (Gly4Ser) 3-Fc measured by the inhibition of HUVEC proliferation was similar to that of D2-9Gly-Fc and D2-9Gly-Ex3 / CH3 (Fig. 6).

Construction D2- (Gly4Ser) 3-Fc was further characterized by Western blotting and compared to D29Gly-Fc (Fig. 9). Both constructions are present mainly in a dimeric form and monomeric forms were detected after the separation of reduced samples.

Example 6

9Gly or VEGF Ex3 paper in Flt-1 (D2) constructions

To investigate the role of the 9Gly linker or the VEGF Ex3 dimerization sequence on the binding of the VEGF soluble receptor, three other constructs were generated: D2-9Gly-CH3, D2-CH3 and D2-Ex3 / CH3 (Fig. 8). The three constructions were generated and like all previous constructions they were also placed under the control of the CMV engine. Its VEGF blocking activity was tested in the HUVEC proliferation assay (Fig. 9).

The HUVEC proliferation assay of proteins containing the CH3 region of IgG1 has shown that D29Gly-CH3 (without Ex3) and D2-Ex3 / CH3 protein (without 9Gly linker) had similar VEGF blocking power compared to D2 protein -9Gly-Ex3 / CH3 parental. However, it seems that D2-CH3 protein is the weakest inhibitor of VEGF among all of them (Fig. 9).

Flt-1 ELISA data from media conditions of transfected 293 cells have similar Flt-1 levels for D2-9Gly-Ex3 / CH3, D2-9Gly-CH3 and D2-Ex3 / CH3 and D2-CH3 (70-90 ng / ml) and slightly higher (�150 ng / ml) for the less active form of D2-CH3. The Western blot of the D2-9Gly-CH3 and D2-CH3 constructs (Fig. 10) demonstrates a predominance of dimeric forms under non-reduced conditions.

Example 7

D2-9Gly-Fc joins VEGF better than all constructions

The VEGF binding assay makes it possible to compare the VEGF-related binding affinities of these soluble VEGF receptors in a cell-free system.

In summary, conditioned media containing known concentrations of soluble receptors (varying in concentrations of 0.29-150 pM) were serially diluted and mixed with 10 pM VEGF. The amount of unbound VEGF was then measured by ELISA. D2-9 Gl-Fc binds to VEGF with greater affinity at receptor concentrations of 0.001 to ~ 0.2 pM than all other constructs. D2-CH3 has the lowest affinity to join VEGF (Fig. 11).

References

Davis-Smyth, et al., EMBO J., 15,1996, 4919 Huston, J. S., et al. (1991) Methods Enzymol. 203, 46-88 Huston, J. S., et al. (1988) Proc. Natl Acad. Sci. USA, 85, 5879-5883. Johnson, S., et al. (1991) Methods Enzymol. 203, 88-98 Karpus, P. A., et al. (1985) Naturwiss., 72, 212-213. Keyt, B. A., et al. (1996) J. Biol. Chem. 271: 5638-5646. Kortt, A. A., et al. (1997) Protein Engng, 10, 423-433. Lee, Y-L., Et al. (1998) Human Gene Therapy, 9, 457-465 Mouz N., et al. (1996) Proc. Natl Acad. Sci. USA, 93, 9414-9419. Nielsen, et al. (1997) Protein Eng., 10, 1 Qiu, H., et al. (1998) J. Biol. Chem. 273: 11173-11176.

Sequence Table

SEQ ID NO.

Clone name Length Type

one

FLT1D29GLYFC 1077 DNA

2

FLT1D29GLYFC 358 Protein

3

FLT1D2DEL9GLYFC 1050 DNA

4

FLT1D2DEL9GLYFC 349 Protein

5

FLT1D29GLYEX3 426 DNA

6

FLT1D29GLYEX3 141 Protein

7

FLT1D29GLYEX3CH3 744 DNA

8

FLT1D29GLYEX3CH3 247 Protein

9

IgG1 HEAVY 1434 DNA

10

IgG1 HEAVY 477 Protein

eleven

 VEGF 648 DNA

12

VEGF 215 Protein

13

VEGF exon 3 (ex3) 14 Protein

14

 FLT-1 5777 DNA

fifteen

FLT-1 1338 Protein

16

 KDR 5830 DNA

17

 KDR 1356 Protein

18

 D2-CH3 675 DNA

19

 D2-CH3 224 Protein

twenty

 D2-EX3-CH3 717 DNA

twenty-one

 D2-EX3-CH3 238 Protein

22

 D2-9GLY-CH3 702 DNA

2. 3

D2-9GLY-CH3 233 Protein

24

 D2 (G4S) 3-Fc 1095 DNA

25

D2 (G4S) 3-Fc 364 Protein

26

 random linker 9 Protein

27

 linker 9 Protein

28

 linker 9 Protein

29

 linker 9 Protein

30

 linker 9 Protein

31

 linker 7 Protein

32

 linker 13 Protein

33

 linker 9 Protein

3. 4

 linker 23 Protein

Preferred embodiments of the present description are described below and referred to as embodiments E1 to E71.

E1. A fusion protein of formula X-Y-Z, where

X comprises a polypeptide selected from the group consisting of an extracellular receptor, a variable region of antibody, a cytokine, a chemokine, and a growth factor; and And it consists essentially of a polypeptide of 5 to 25 amino acid residues, and

Z is a CH3 region of an IgG heavy chain molecule E2 The E1 fusion protein wherein X comprises an extracellular receptor and said receptor is selected from the group consisting of a tyrosine kinase receptor and a serine threonine kinase receptor.

E3 The E1 fusion protein wherein X comprises an extracellular receptor and said receptor is a receptor of the VEGF E4 The fusion protein of E1 where X is the IgG-like domain 2 of VEGF-R1 (FLT-1).

E5. The E1 fusion protein where the Y polypeptide is flexible. E6 The E1 fusion protein wherein the Y polypeptide is selected from the group consisting of gly9 (SEQ ID NO: 27), glu9 (SEQ ID NO: 28), ser9 (SEQ ID NO: 29), gly5cyspro2cys (SEQ ID NO: 30), (gly4ser) 3 (SEQ ID NO: 31), SerCysValProLeuMetArgCysGlyGlyCysCysAsn (SEQ ID NO: 32), Pro Ser Cys Val Pro Leu Met Arg Cys Gly Gly Cys Cys Asn (SEQ ID NO: 13), Gly-Asp-Leu-Ile-Tyr-Arg-Asn-Gln-Lys (SEQ ID NO: 26), and Gly9ProSerCysValProLeuMetArgCysGlyGlyCysCysAsn (SEQ ID NO: 34).

E7 The E1 fusion protein where Z is a CH3 region of IgG1. E8 The E7 fusion protein where Z is a CH3 region of IgG2. E9. A composition comprising the E1 fusion protein that further comprises one or more excipients or

pharmaceutically acceptable vehicles. E10 The composition of E9 wherein said composition is a liquid formulation. E11 The composition of E9 wherein said composition is a lyophilized formulation. E12 A composition comprising one or more E1 fusion proteins, wherein said composition

It comprises multimers of said fusion proteins.

E13 The composition of E12, wherein said composition consists essentially of a single species of protein of fusion that forms homomultimeros. E14 The composition of E12, wherein said composition consists essentially of fusion proteins, wherein

the X residues in said fusion proteins in the composition are heterogeneous.

E15 The composition of E12 wherein the remainder X is an extracellular receptor, and said receptor is selected from the group consisting of a tyrosine kinase receptor and a serine threonine kinase receptor. E16 The composition of E12 wherein the remainder X is an extracellular receptor, and said receptor is a receptor of the

VEGF or a portion thereof. E17. The composition of E12 wherein the remainder X is domain 2 of the IgG type of VEGF-R1 (FLT-1). E18 The composition of E12 wherein Y is a flexible polypeptide. E19. The composition of E12 wherein Y is a polypeptide selected from the group consisting of gly9 (SEQ ID NO:

27), glug (SEQ ID NO: 28), ser9 (SEQ ID NO: 29), gly5cyspro2Cys (SEQ ID NO: 30), (gly4ser) 3 (SEQ ID NO: 31), SerCysValProLeuMetArgCysGlyGlyCysCysAsn (SEQ ID NO: 32), Pro Ser Cys Val Pro Leu Met Arg Cys Gly Gly Cys Cys Asn (SEQ ID NO: 13), Gly-Asp-Leu-Ile-Tyr-Arg-Asn-Gln-Lys (SEQ ID NO: 26), and Gly9ProSerCysValProLeuMetArgCysGlyGlyCysCysAsn (SEQ ID NO: 34).

E20 The composition of E12 wherein Z is a CH3 region and said region is a CH3 region of IgG1.

E21 The composition of E12 which further comprises one or more pharmaceutically excipients or vehicles acceptable. E22 The composition of E21 wherein said composition is a liquid formulation. E23 The composition of E21 wherein said composition is a lyophilized formulation. E24 A polypeptide of formula X-Y-Z, wherein X comprises a polypeptide selected from the group consisting of an extracellular receptor, a variable region of

antibody, a cytokine, a chemokine, and a growth factor;

And it consists essentially of a linker moiety that provides the spatial separation of 5-25 moieties amino acids; Y Z is a CH3 region of an IgG heavy chain molecule.

E25 The E24 polypeptide wherein the linker moiety is an oligomer having 10-100 ethylene glycol monomer moieties

E26 A fusion protein of formula X-Y-Z, where X comprises a polypeptide selected from the group consisting of an extracellular receptor, a variable region of antibody, a cytokine, a chemokine, and a growth factor; and

And it consists essentially of a polypeptide of 5 to 25 amino acid residues, and Z is an Fc portion of an antibody molecule. E27 The E26 fusion protein, where Fc is an IgG Fc. E28 The fusion protein of E27, where the IgG Fc is IgG1 Fc. E29 A composition comprising the E26 fusion protein that further comprises one or more excipients

or pharmaceutically acceptable vehicles. E30 The composition of E29 wherein Z is an Fc portion of an antibody molecule that is an IgG Fc. E31 The composition of E29 wherein Z is an Fc portion of an antibody molecule that is an Fc of IgG1. E32 A fusion protein of formula X-Y-Z, where X comprises a polypeptide selected from the group consisting of an extracellular receptor, a variable region of

antibody, a cytokine, a chemokine, and a growth factor; and

And it consists essentially of a linker moiety that provides the spatial separation of 5-25 moieties amino acids, and Z is an Fc portion of an antibody molecule. E33 The E32 fusion protein, where Fc is an IgG Fc. E34 The E33 fusion protein, where the IgG Fc is IgG1 Fc. E35 The E32 fusion protein wherein the linker moiety is an oligomer that has 10-100 monomeric moieties

of ethylene glycol. E36 A composition comprising the E32 fusion protein that further comprises one or more excipients

or pharmaceutically acceptable vehicles. E37 The composition of E36 wherein Z is an Fc portion of an antibody molecule that is an IgG Fc. E38 The composition of E36 wherein Z is an Fc portion of an antibody molecule that is an Fc of IgG1. E39 A method for multimerizing an X polypeptide comprising: binding an X polypeptide to a Z polypeptide by a Y polypeptide to form an XYZ polypeptide, wherein X comprises a polypeptide selected from the group consisting of an extracellular receptor, a variable region of

antibody, a cytokine, a chemokine, and a growth factor Y consists essentially of a polypeptide 5 to 25 amino acid residues, and Z is a CH3 region of an IgG heavy chain molecule;

whereby the XYZ polypeptide is multimerized. E40 The method of E39 wherein the binding step comprises constructing a nucleic acid molecule that encodes each of X, Y, and Z as a single open reading frame, wherein said XYZ polypeptide is expressed from the construction of nucleic acid in a host cell.

E41 A method for multimerizing an X polypeptide comprising: linking an X polypeptide to a Z polypeptide by a Y polypeptide to form an XYZ polypeptide, wherein X comprises a polypeptide selected from the group consisting of an extracellular receptor, a variable region of

antibody, a cytokine, a chemokine, and a growth factor;

And it consists essentially of a linker moiety that provides the spatial separation of 5-25 moieties amino acids, and Z is a CH3 region of an IgG heavy chain molecule; whereby the XYZ polypeptide is multimerized.

E42 The E41 method where the linker moiety essentially consists of an oligomer of 10-100 residues ethylene glycol monomers. E43 A nucleic acid molecule that encodes a fusion protein according to E1. E44 A nucleic acid molecule that encodes a fusion protein according to E26.

E45 The nucleic acid of E43 or 44 where X is the 2 domain of type Ig of VEGF-R1 (Flt-1). E46 The E43 nucleic acid where X is the 2 Ig domain of VEGF-R1 (Flt-1) and the fusion protein it comprises a sequence selected from the group consisting of SEQ ID NO: 8, 21, and 23.

E47 The E44 nucleic acid where X is the 2 Ig domain of VEGF-R1 (Flt-1) and the fusion protein It comprises a sequence selected from the group consisting of SEQ ID NO: 2 and 25.

E48 The fusion protein of E1 or 26 where X is the 2 domain of type Ig of VEGF-R1 (Flt-1). E49 The E1 fusion protein where X is the 2 Ig domain of VEGF-R1 (Flt-1) and the fusion protein it comprises a sequence selected from the group consisting of SEQ ID NO: 8, 21, and 23.

E50 The E26 fusion protein where X is the VEGF-R1 Ig-like domain 2 (Flt-1) and the fusion protein It comprises a sequence selected from the group consisting of SEQ ID NO: 2 and 25. E51 A mammalian cell comprising the nucleic acid of E43 or 44.

E52 The mammalian cell of E51 which is a human cell. E53 The E52 mammalian cell that is selected from the group consisting of a fibroblast, a hepatocyte, a endothelial cell, a keratinocyte, a hematopoietic cell, a synoviocyte, an epithelial cell, a cell retinal, and a stem cell.

E54 An in vitro method comprising:

supplying the E43 or 44 nucleic acid to an isolated mammalian cell to form a cell that expresses said fusion protein. E55 The E54 method wherein the fusion protein comprises a sequence selected from the group that

consists of SEQ ID NO: 2, 8, 21, 23, and 25. E56 The method of E54 further comprising: supplying the cell that expresses said fusion protein to a mammal. E57 A method comprising: supply the mammalian cell of E51 to a mammal. E58 A method comprising: supplying the E43 or 44 nucleic acid to a mammal, whereby said fusion protein is expressed in the

mammal.

E59 The E58 method wherein the fusion protein comprises a sequence selected from the group that consists of SEQ ID NO: 2, 8, 21, 23, and 25. E60 The E57 method where the mammal has age-related wet macular degeneration or

proliferative diabetic retinopathy. E61 The E58 method where the mammal has cancer. E62 The E58 method where the mammal has rheumatoid arthritis. E63 The E58 method where the mammal has asthma. E64 The E58 method where the mammal has osteoarthritis. E65 A method comprising: deliver the fusion protein of E1 or 26 to a mammal. E66 The E65 method where the mammal has age-related wet macular degeneration or

proliferative diabetic retinopathy. E67 The E65 method where the mammal has cancer. E68 The E65 method where the mammal has rheumatoid arthritis. E69 The method of E65 where the mammal has asthma.

E70 The E65 method where the mammal has osteoarthritis.

E71 The method of E65 wherein the fusion protein comprises a sequence selected from the group consisting of SEQ ID NO: 2, 8, 21, 23, and 25.

5 Sequence listing

<110> Genzyme Corporation

<120> MULTIMERICAL CONSTRUCTIONS

<130> P30465EP-PCT

<140> EP05810409.2 10 <141> 2005-09-13

<150> 60/658209

<151>

<150> 60/608887

<151> 15 <160> 34

<170> FastSEQ for Windows version 4.0

<210> 1

<211> 1077

<212> DNA 20 <213> Artificial sequence

<220>

<223> Recombinant fusion protein or sequence encoding it

<400> 1

25 <210> 2

<211> 358

<212> PRT

<213> Artificial sequence

<220> 30 <223> Recombinant fusion protein or sequence encoding it

<400> 2

<210> 3 5 <211> 1050

<212> DNA

<213> Artificial sequence

<220>

<223> Recombinant fusion protein or sequence encoding it 10 <400> 3

<210> 4

<211> 349

<212> PRT

<213> Artificial sequence

<220>

<223> Recombinant fusion protein or sequence encoding it

<400> 4 <210> 5

<211> 426

<212> DNA

<213> Artificial sequence

<220>

<223> Recombinant fusion protein or sequence encoding it

<400> 5

<210> 6

<211> 141

<212> PRT

<213> Artificial sequence

<220>

<223> Recombinant fusion protein or sequence encoding it

<400> 6

<210> 7

<213> Artificial sequence

<220>

<223> Recombinant fusion protein or sequence encoding it

<400> 7

<210> 8

<211> 247

<212>

PRT 20 <213> Artificial sequence

<220>

<223> Recombinant fusion protein or sequence encoding it

<400> 8

<210> 9

<211> 1434

<212> DNA

<213> Homo sapiens

<400> 9

<210> 10

<211> 477

<212> PRT

<213> Homo sapiens

<400> 10

<210> 11

<211> 648

<212> DNA

<213> Homo sapiens

<400> 11

<210> 12

<211> 215

<212>

PRT 10 <213> Homo sapiens

<400> 12

<212> PRT

<213> Homo sapiens

<400> 13

<210> 14

<211> 5777

<212> DNA

<213> Homo sapiens

<400> 14

<210> 15

<211> 1338

<212> PRT

<213> Homo sapiens

<220>

<221> DOMAIN

<222> (235) ... (336)

<223> domain 3

<400> 15

<210> 16

<211> 5830

<212> DNA

<213> Homo sapiens

<400> 16

<210> 17

<211> 1356

<212> PRT

<213> Homo sapiens

<400> 17

<210> 18

<211> 675

<212> DNA

<213> Artificial sequence

<220>

<223> Recombinant fusion protein or sequence encoding it

<400> 18

10 <210> 19

<211> 224

<212> PRT

<213> Artificial sequence

<220>

<223> Recombinant fusion protein or sequence encoding it

<400> 19 <210> 20

<211> 717

<212> DNA

<213> Artificial sequence

<220>

<223> Recombinant fusion protein or sequence encoding it

<400> 20

10 <210> 21

<211> 238

<212> PRT

<213> Artificial sequence

<220>

<223> Recombinant fusion protein or sequence encoding it

<400> 21 <210> 22

<211> 702

<212> DNA

<213> Artificial sequence

<220>

<223> Recombinant fusion protein or sequence encoding it

<400> 22

10 <210> 23

<211> 233

<212> PRT

<213> Artificial sequence

<220>

<223> Recombinant fusion protein or sequence encoding it

<400> 23 <210> 24

<211> 1095

<212> DNA

<213> Artificial sequence

<220>

<223> Recombinant fusion protein or sequence encoding it

<400> 24

10 <210> 25

<211> 364

<212> PRT

<213> Artificial sequence

<220>

<223> Recombinant fusion protein or sequence encoding it

<400> 25

<210> 26

<211> 9

<212> PRT

<213> Artificial sequence 10 <220>

<223> fusion protein linker

<400> 26

<210> 27

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> linker moiety for fusion proteins

<400> 27

<210> 28

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> linker moiety for fusion proteins

<400> 28

<210> 29

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> linker moiety for fusion proteins

<400> 29

<210> 30

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> linker moiety for fusion proteins

<400> 30

<210> 31

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> linker moiety for fusion proteins

<400> 31

<210> 32

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> linker moiety for fusion proteins

<400> 32

<210> 33

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> linker moiety for fusion proteins

<400> 33

<210> 34

<211> 23

<212> PRT

<213> Artificial sequence

<220>

<223> linker moiety for fusion proteins

<400> 34

Claims (19)

one.

 A fusion protein of formula X-Y-Z, in which: X comprises a portion of the VEGF receptor; And it is a linker moiety; Y Z is a multimerization domain, wherein the linker moiety Y is a polypeptide of 5-50 amino acid residues that has a flexibility above

of the average as determined by the prediction method of flexibility of Karpus and Schultz, 1985, Naturwiss, 72: 212-213, and where said fusion protein binds to VEGF when it is multimerized.

2.

 The fusion protein of claim 1, wherein the linker moiety Y is selected from the compound group by Gly9 (SEQ ID No. 27), Glu9 (SEQ ID No. 28), Ser9 (SEQ ID No. 29), Gly5CysPro2Cys (SEQ ID No. 30), (Gly4Ser) 3 (SEQ ID No. 31), SerCysValProLeuMetArgCysGlyGlyCysCysAsn (SEQ ID No. 32), Pro Ser Cys Val Pro Leu Met Arg Cys Gly Gly Cys Cys Asn (SEQ ID No. 13),

Gly-Asp-Leu-Ile-Tyr-Arg-Asn-Gln-Lys (SEQ ID No. 26), and Gly9ProSerCysValProLeuMetArgCysGlyGlyCysCysAsn (SEQ ID No. 34).

3.

The fusion protein of claim 1 or claim 2, wherein the multimerization domain Z causes the migration of at least 50% of the monomeric fusion proteins in a non-naturalizing polyacrylamide gel at a rate that is appropriate for a multimer of fusion protein.

Four.

The fusion protein of claim 1 or claim 2, wherein the multimerization domain Z is a fragment of an IgG heavy chain.

5.

The fusion protein of claim 4, wherein the multimerization domain Z is a sequence of an Fc portion of an IgG1 or IgG2 heavy chain.

6.

The fusion protein of claim 1 or claim 2, wherein the multimerization domain Z is a CH3 region of an IgG heavy chain or is an Fc portion of an antibody molecule.

7.

The fusion protein of any one of claims 1 to 6, wherein X comprises domain 2 of IgG type of VEGF-R1.

8.

The fusion protein of claim 7, wherein X comprises VEGF-R1 IgG type 2 domain but lacks VEGF-R1 Ig type 1 and 3 domains.

9.

The fusion protein of claim 8, wherein X is the IgG-like domain 2 of VEGF-R1.

10.

A composition comprising the fusion protein of any one of claims 1 to 9 and one or more pharmaceutically acceptable carriers.

eleven.

A method for multimerizing an X polypeptide, the method comprising joining the X polypeptide to a Z polypeptide by a Y polypeptide to form an X-Y-Z polypeptide, wherein:

X comprises a portion of a VEGF receiver; And it is a linker moiety; yZ is a multimerization domain, where the linker moiety Y is a polypeptide of 5-50 amino acid residues that has an above-average flexibility as determined by the method of predicting flexibility of Karpus and Schultz, 1985, Naturwiss, 72: 212-213, and where said fusion protein binds to VEGF when multimerized.

12.

The method of claim 11, wherein the binding step comprises constructing an acidic molecule that encodes polypeptides X, Y and Z as a single open reading frame, wherein said XYZ polypeptide is expressed from said acid molecule nucleic in a host cell.

13.

A nucleic acid molecule encoding the fusion protein of any one of claims 1 to

9. 14. The nucleic acid molecule of claim 13, which encodes a fusion protein of claim 1, wherein the fusion protein comprises a sequence that is selected from the group consisting of SEQ ID NOs: 2, 8, 21, 23 and 25 15. A vector comprising the nucleic acid molecule of claim 13 or 14. 16. The vector of claim 15, wherein the vector is an adeno-associated viral vector. 17. A mammalian cell comprising the nucleic acid molecule of claim 13 or 14 or the vector of claim 15 or 16. 18. An in vitro method comprising delivering the nucleic acid of claim 13 or 14 or the vector of claims 15 or 16 to a mammalian cell to produce a cell expressing said fusion protein. 19. The fusion protein of any one of claims 1 to 9, the nucleic acid molecule of claim 13 or 14, the vector of claim 15 or 16, or the mammalian cell of claim 17, for use in the treatment of a mammal. 20. The fusion protein, nucleic acid molecule, vector or mammalian cell for use according to claim 20 19, where the mammal has age-related wet macular degeneration, proliferative diabetic retinopathy, cancer, rheumatoid arthritis, asthma, or osteoarthritis.